Expression of vitreoscilla hemoglobin in Aurantiochytrium sp. enhancesthe production of fatty acids and astaxanthin

Abstract

Heterotrophic fermentation is a widely used means in the biotechnological, pharmaceutical and food industries for the large-scale production of desired products. However, two problems are often encountered during its application: the limitation of dissolved oxygen level in high cell density cultures, and the high cost of the carbon substrates for supporting growth. In this study these issues are solved through the expression of the hemoglobin gene from Vitreoscilla (VHb), which is known to be able to facilitate intracellular oxygen delivery, and the use of low-cost waste carbon sources for fermentation, respectively. Aurantiochytrium sp. MP4 (non-pigmented) and SK4 (pigmented) were chosen for the present study because of members of this genus has been consideredas potential producers of biodiesel as well asastaxanthin in recent years.

VHb gene was successfully expressed in Aurantiochytrium sp. MP4 and SK4, and three transformants of MP4 (TMP4-VHb1, TMP4-VHb10 and TMP4-VHb24) and one transformant of SK4 (TSK4-VHb2) were obtained, respectively. TMP4-VHb24 andTSK4-VHb2 were selected for further study. It was found that VHb expression led to higher maximum biomass under microaerobic conditions and improved oxygen utilization in high cell density fermentation. Moreover, under aerobic conditions, there was an increase of total cellular fatty acid content by 10% and 44%forTMP4-VHb24 and TSK4-VHb2,respectively, and the pigmented strain TSK4-VHb2 produced 9-fold higher astaxanthin content than the control (i.e., SK4), showing that heterologous expression of VHb as a promising approach to improving growth or secondary metabolite biosynthesis in the fermentation process.

The mechanism underlying the effects of VHb expression was further investigated using TMP4-VHb24. Expression of VHb led to a higher adenylate energy charge and hence higher metabolically available energy in the cells, especially at the late exponential phase. In addition, VHb expression promoted the rate of consumption of NADH during the period from the late exponential phase to the stationary phase, in which NADH carried electrons from the oxidation of carbon substrates in energy pathways for ATP synthesis. VHb also increased the resistance of the host cells to nitrosative stress, but not to oxidative stress.

In terms of utilization of cheap carbon substrates, TMP4-VHb24 and TSK4-VHb2were found to grow well on the waste carbon sources including cane molasses and crude glycerol. Under such conditions, however, the contents of fatty acids and astaxanthin were generally lowered compared to the use of glucose-based media. Therefore, the growth and product synthesis in TMP4-VHb24 was further optimized using the response surface methodology, and it was found to be effective. In conclusion, the expression of VHb in Aurantiochytrium sp. MP4 and SK4 was effective in relieving limitation on the growth due to low oxygen availability at high cell density cultivation, while increasing the cellular astaxanthin and fatty acid biosyntheses under aerobic conditions. Also, the VHb-expressing strains were able to utilize low-cost waste carbon sources, and the response surface methodology could be employed to optimize fermentation effectively in order to lower the cost of fermentation.